The food and beverage industries, as well as other industries such as the cosmetic industry and the health care industry, routinely use flavors to enhance their products' appeal. Natural flavors are especially desired by consumers for reasons of health, fitness, safety, aesthetics, etc. Worldwide consumer demand for natural flavors thus continues to increase. Natural flavors or natural flavorings have been defined at 21 C.F.R. .sctn.101.22 as the essential oil, oleoresin, essence or extractive, protein hydrolysate, distillate, or any products of roasting, heating or enzymolysis which contains the flavoring constituents derived from a spice, fruit or fruit juice, vegetable or vegetable juice, edible yeast, herb, bark, bud, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products or fermentation products thereof, whose significant function in food is flavoring rather than nutrition.
One such flavor is nootkatone (4,4a,5,6,7,8-hexahydro-6-isopropenyl-4,4a-dimethyl-2(3II)-naphtalenone). Nootkatone is an important flavor constituent of grapefruit and is used commercially to flavor soft drinks and other beverages, as well as being used in perfumery. The conventional method for nootkatone preparation is by oxidation of valencene. However, nootkatone prepared by standard chemical oxidation methods cannot be considered a natural flavorant and thus has less commercial desirability. Additionally, the starting material valencene is expensive and thus methods that consume valencene are less commercially acceptable. Because of these drawbacks, there is still a need for commercially feasible methods to prepare natural nootkatone.
Several methods exist for the production of nootkatone, but each has limitations. For example, U.S. Pat. No. 5,847,226 discloses nootkatone production by oxidizing valencene to nootkatone, nootkatol or mixtures of nootkatone and nootkatol in the presence of an unsaturated fatty acid hydroperoxide. However, in the '226 patent the fatty acid hydroperoxide is generated by autooxidation, photooxidation, chemically catalyzed oxidation or by enzymatic oxidation using lipoxygenase, and then the fatty acid hydroperoxide catalyzes the autooxidation of valencene. Such an autooxidization is generally not a selective process. Previous attempts to use microbial or enzymatic transformations to prepare nootkatone have generally resulted in low yields. For example, Drawert et al. (1984) reported the biotransformation of valencene to nootkatone in cell suspension cultures but the starting level of valencene was too low to be practical. del Rio et al. (1991) studied the accumulation of nootkatone and valencene by callus cultures of Citrus spp. The level of nootkatone in nine-month old callus cultures was between 16-160 .mu.g/100 g fresh weight. More recently, a Japanese patent disclosed biotransformation of valencene to nootkatone using a selected microbial fermentation, but the overall yield was low. In shake flask experiments with 50 ml working volume, only 2.5 mg of nootkatone was obtained from 500 mg of starting valencene (Okuda et al., 1994).
Thus, a commercially viable method to produce nootkatone utilizing laccase would be desirable.